title: Compromised repair of clustered DNA damage in the human acute lymphoblastic leukemia MSH2-deficient NALM-6 cells.
abstract: Ionizing radiation (IR) induces two classes of complex DNA damage, double-strand breaks (DSBs) and non-DSB bi-stranded oxidative clustered DNA lesions (OCDLs). OCDLs may consist of single strand breaks (SSBs), oxidized purines/pyrimidines and abasic sites within 5-10bp. These significant biological lesions are hypothesized to challenge the repair machinery and carry a high mutagenic potential. MSH2, a classical DNA mismatch repair protein, has been also implicated in other repair pathways associated with DSB and base lesion processing. MSH2 mutations have been identified in acute lymphoblastic leukemia (ALL) patients as well as in other types of cancers. Our research model involves two precursors B (pre-B) ALL human cell lines, NALM-6 cells, homozygous null for MSH2, and wild type 697 cells. Using a modified version of neutral and alkaline single cell gel electrophoresis (SCGE) with Escherichia coli repair enzymes as damage probes, the processing capacity of single strand breaks (SSBs), DSBs and OCDLs was assessed in NALM-6 and 697 cells exposed to a radiotherapy relevant gamma-ray dose of 5Gy. Using reverse transcriptase PCR and Western blotting we verified the complete lack of expression of MSH2 in the NALM-6 cells at the transcriptional and translational level. No differences were measured between NALM-6 and 697 cells in the induction levels of SSBs, DSBs and OCDLs after exposure to gamma-rays. However, 697 cells repaired each lesion more efficiently with significant differences observed after 1-3h post-irradiation. Lastly, our results indicate a significantly higher population of apoptotic 697 cells compared to NALM-6 cells 6-24h post-irradiation. Our studies suggest that MSH2 is probably involved in the processing of the biologically significant clustered DNA damages as well as the execution of apoptosis induced by ionizing radiation.
authors.affiliation: Department of Biology, Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, NC 27858, USA.
authors.forename: Stewart M
authors.initials: SM
authors.lastname: Holt
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Scemama
authors.forename: Mihalis I
authors.initials: MI
authors.lastname: Panayiotidis
authors.forename: Alexandros G
authors.initials: AG
authors.lastname: Georgakilas
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D045744:Cell Line, Tumor; D004249:DNA Damage / Q000235:genetics* / Q000528:radiation effects; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D005720:Gamma Rays; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008954:Models, Biological; D005810:Multigene Family / Q000528:radiation effects; D051718:MutS Homolog 2 Protein / Q000235:genetics* / Q000502:physiology; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics* / Q000473:pathology; D011879:Radiotherapy Dosage
chemical_list: D006861:Hydrogen Peroxide; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein